CN110172430A - One plant of mulberry enterobacteria and its application in degradation Diacloden - Google Patents
One plant of mulberry enterobacteria and its application in degradation Diacloden Download PDFInfo
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- CN110172430A CN110172430A CN201910525694.3A CN201910525694A CN110172430A CN 110172430 A CN110172430 A CN 110172430A CN 201910525694 A CN201910525694 A CN 201910525694A CN 110172430 A CN110172430 A CN 110172430A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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Abstract
The present invention relates to the mulberry enterobacteria (Enterobacter mori) that one plant of deposit number is CGMCC No.16236 and its applications in degradation Diacloden；The mulberry enterobacteria can be prepared into microbial inoculum, applied to the reparation of soil source Diacloden residual contamination in leaf vegetables planting system, and reduce the Diacloden residual of leaf vegetables blade, and promote plant strain growth；The mulberry Enterobacter can be colonized in vegetable plant strain in Soil Probiotics for a long time, under the premise of not influencing quality of vegetable, efficiently reduce Diacloden residual, realize green safe production.
Description
Technical field
The present invention relates to one plant of mulberry enterobacteria (Enterobacter mori) and its applications in degradation Diacloden, i.e.,
For in leaf vegetables planting system the Soil Utilization of the soil source plasticiser Diacloden residual contamination mulberry enterobacteria prepare microbial inoculum simultaneously
It is applied, belongs to microbiology and field of food safety.
Technical background
The development of vegetables industry optimizes and revises China's crop mix, growth of agricultural efficiency, increasing peasant income, expands foreign exchange earning
Etc. have irreplaceable role.Vegetables breeding time is short, and growth is fast, and multiple crop index is high, is highly prone in process of production
The harm of pest and disease damage.According to incompletely statistics, the insect pest of China vegetables has more than 200, every year need to be using a large amount of pesticides to ensure vegetables
From or it is few influenced by insect pest, but environmental pollution is caused in certain degree for a large amount of uses of pesticide and food safety is hidden
Suffer from.Other than directly spraying the pesticide on crop, crop can also absorb pesticide residue from contaminated soil and water, be made
The pesticide residue that object absorbs may be entered eventually into food chain and be threatened to the health of humans and animals.Therefore environment is eliminated
In or the intracorporal pesticide residue of crop have vital effect to ensuring food safety.It traditional physics and is chemically removed
Soil and water pollutant are not only costly, and certain destructiveness can be also caused to environment.
Anabasine insecticide is a kind of novel pesticide mainly developed in recent decades.The insecticides dosage is few,
Persistence is strong, to the high neurotoxicity of most of arthropods and unique selectively acting mechanism, in the time less than 20 years
In, develop as the most popular insecticides in the whole world, imidacloprid and Diacloden be in the insecticides two it is main
Kind.Anabasine insecticide both can be used as cauline leaf process, it can also be used to soil and seeds processing.However, anabasine desinsection
The a large amount of influences using to environment and non-target organism of agent worldwide cause the highest attention of various countries, have a large amount of
Document report anabasine insecticide to the Pollinating Insects such as honeybee and earthworm have high poison, can also cause bee colony collapse imbalance
Disease；Moreover, this insecticides also has high genotoxicity to birds；It influences the respiration of muroid and liver is caused to damage
Wound；Even result in the decline of bio-diversity.Anabasine insecticide effective component 8 used are registered in China, are related to correlation
Product 1729, be the insecticides that China is currently most widely used on vegetable and fruit and cereal crops.
[1] Diacloden (thiamethoxam) is efficient, the safety, high selection researched and developed by Novartis Co., Ltd, Switzerland in 1991
Property, low toxicity second generation novel nicotinamide insecticide, increased with chlorine thiazole ring and broad-spectrum action and improve activity, polarity is strong, dissolution
Property it is high, systemic action is strong, spraying, soil pouring root, seed treatment for cauline leaf, is administered rear blade or root system and is passed rapidly by interior suction
It is defeated to arrive each position of plant, the pests such as aphid, leaf miner can be effectively prevented.It can there are the thiophene worms of high concentration in soil and surface water
Piperazine.And the residual of high concentration is detected in honey and corn and the pollen of sunflower, brown rice, oat, lemon.Existing a large amount of text
It offers and reports Diacloden and have high poison to the Pollinating Insects such as honeybee and earthworm, can also causing bee colony collapse imbalance disease, (" Wu Chi is several
Anabasine insecticide to Apis mellifera toxic effect study [D] Agricultural University Of Hunan, 2015 ", " Girolami V, Marzaro M,
Vivan L, et al.Aerial powdering of bees inside mobile cages and the extent of
neonicotinoid cloud surrounding corn drillers[J].Journal of Applied
Entomology, 2013,137 (1-2): 35-44 ", " Garry Codling, Yahya Al Naggar.Neonicotinoid
insecticides in pollen,honey and adult bees in colonies of the European honey
bee(Apis mellifera L.)in Egypt[J].2017”、“Jeroen P van der Sluijs1,Noa Simon-
Delso1,Dave Goulson2,Neonicotinoids,bee disorders and the sustainability of
pollinator services[J].2013")；Moreover, this insecticides also has high genotoxicity to birds；It influences
The respiration of muroid and cause hepar damnification；Even result in decline (" Wenchao the Han a, Ying of bio-diversity
Tian a,b,*,Xiaoming Shen a,**Human exposure to neonicotinoid insecticides and
the evaluation of their potential toxicity:An overview,2018”、“Alzbeta Stara
a,b,Roberto Bellinvia b,Josef Velisek a,Alzbeta Strouhova a,Antonin Kouba a,
Caterina Faggio,Acute exposure of common yabby(Cherax destructor)to the
neonicotinoid pesticide,2019").The half-life period of Diacloden in the soil is 50 days (7-72 days).In soil
Diacloden is mainly by microbial degradation.
Mulberry enterobacteria (covers handsome honor etc., Guangxi mulberry tree bacterial wilt germ biology other than report is pathogenic bacteria at present
Characteristic and pesticide control screen [J] Guangxi plant protection, 2015,28 (3): 1-7.), in terms of yet there are no degradation of contaminant or pesticide
Document report, there are no mulberry Enterobacter degradation anabasine insecticide report.
Summary of the invention
The technical problem to be solved in the present invention is to provide one plant of mulberry enterobacteria (Enterobacter mori), which can
It degrades for the main Diacloden of soil, and can further be prepared as renovation agent, can effectively degrade and remain on plantation soil
In Diacloden pollution, and then reduce its absorption transfer into leaf vegetables, while influencing the normal growth of leaf vegetables plant, and not
Introduce other pollution sources.
To achieve the above object, present invention firstly provides the mulberry enterobacterias that one plant of deposit number is CGMCC NO:16236
(Enterobacter mori), for the strain isolation from perennial deciduous tree or shrub plant mulberry tree body, bacterial strain is in rod
Shape, not endogenous spore, Enterobacter；The bacterial strain majority is harmless to animals and plants, which is named as TMX-13 certainly by applicant, and
In the preservation on the 8th of August in 2018 to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, postcode: 100101, deposit number is CGMCC No.16236, and classification naming is mulberry
Enterobacteria (Enterobacter mori).
Secondly, the present invention provides the mulberry enterobacterias that above-mentioned deposit number is CGMCC NO:16236 in degradation Diacloden
Using the especially application in degrade soil or leaf vegetables plant body in Diacloden residual.
Further, application of the above-mentioned bacterial strains CGMCC NO:16236 in leaf vegetables plant body in Diacloden residual refers to, incites somebody to action
It is 10 that the mulberry enterobacteria that deposit number is CGMCC NO:16236, which is prepared into bacterial content,7-8The bacterium solution of CFU/ml (OD value is 1), to leaf
Dish plant carries out the processing of leaching root for 24 hours, the Diacloden to edible site residues in leaf vegetables plant of degrading.
Further, application of the above-mentioned bacterial strains CGMCC NO:16236 in degradation soil Diacloden residual refers to, by preservation
It is 10 that number mulberry enterobacteria for being CGMCC NO:16236, which is prepared into bacterial content,7-8The bacterium solution of CFU/ml (OD value is 1), plants leaf vegetables
Strain ground position carries out spraying processing (60ml/m2), the Diacloden to edible site residues in leaf vegetables plant of degrading.
The present invention also provides the mulberry enterobacterias that above-mentioned deposit number is CGMCC NO:16236 to promote leaf vegetables plant strain growth,
Application especially in the growth of leaf vegetables plant root.The application refers to that it is 10 that the mulberry enterobacteria, which is prepared into bacterial content,7- 8CFU/ml (bacterium solution OD600=1) microbial inoculum carries out leaching root processing to leaf vegetables plant, to promote leaf vegetables plant root to grow.
Compared with prior art, mulberry enterobacteria (Enterobacter mori) provided by the invention separates from plant, belongs to
In endophyte of plant mulberry enterobacteria (Enterobacter mori), which can be dropped for pesticide Diacloden in plant
Solution, and it can be further prepared as renovation agent, and then reduce its absorption transfer into leaf vegetables, the normal life of leaf vegetables plant is not influenced
It is long, other pollution sources are not introduced.
Attached drawing subordinate list explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of mulberry enterobacteria (Enterobacter mori) TMX-13.
Fig. 2 is degradation function verification result schematic diagram of the mulberry enterobacteria TMX-13 to Diacloden.
Fig. 3 is that the microbial bacterial agent soaks piece-root grafting bacterium and foliage-spray connects bacterium processing and pollutes to Diacloden in leaf vegetables body is reduced
Effect diagram.
Fig. 4 is effect diagram of the microbial bacterial agent to leaf vegetables vegetables growth-promoting.
Table 1 is phenotypic data of the microbial bacterial agent to leaf vegetables vegetables growth-promoting.
Specific embodiment
Culture medium involved in embodiment:
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.0；
Inorganic salt liquid culture medium: MgSO4·7H2O(0.4g),FeSO4·7H2O(0.2g),K2HPO4(0.2g),(NH4)
2SO4(0.2g),and CaSO4, (0.08g), deionized water 1L, pH 7.0~7.2；
Seed culture medium: K2HPO4(4.8g),KH2PO4(3.5g),(NH4)2SO4(2g),MgCl2(0.16g),CaCl2
(0.02g),NaMoO4.2H2O(0.0024g),FeCl3(0.0018g),MnCl2.2H2O (0.0015g), PH=7.0 are added water to
1L；
Inorganic salts solid medium: 20g/L agar is added in inorganic salt liquid culture medium；
Strain activation and culture base: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.0；
Inorganic salt liquid culture medium: MgSO4·7H2O(0.4g),FeSO4·7H2O(0.2g),K2HPO4(0.2g),
(NH4)2SO4(0.2g),and CaSO4, (0.08g), deionized water 1L, pH 7.0~7.2；
Reagent involved in following embodiment is unless stated otherwise commercial channel purchase.
The separation identification of 1 mulberry enterobacteria TMX-13 of embodiment and degradation function are verified
1, bacterial strain Enterobacter mori sp.TMX-13 separation identification
Applicant has experimental field artificially sprayed thiophene to mulberry tree from Jiangsu Province Agriculture Science Institute the south in the acquisition of in September, 2016
Then the root of the mulberry tree plant of worm piperazine carries out surface sterilization to Mulberry Roots, is ground into juice, it is unique for being coated on containing Diacloden
On the inorganic salts solid medium of carbon nitrogen source, screening obtains one plant of endophyte of plant for having Diacloden degradation characteristic.Applicant
By the bacterium from TMX-13 is named as, electron microscopic picture is as shown in Figure 1, the bacterial strain is in rod shape, no endogenous spore.
TMX-13 bacterial strain, which is compared, by physio-biochemical characteristics and 16SrDNA conserved sequence is accredited as mulberry enterobacteria
(Enterobacter mori), applicant are general to China Committee for Culture Collection of Microorganisms in the preservation on the 8th of August in 2018
Logical microorganism center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode: 100101, deposit number is
CGMCC No.16236, classification naming are mulberry enterobacteria (Enterobacter mori).
2, Enterobacter mori sp.TMX-13 strains for degrading DBP functional verification
First with LB culture medium be incubated overnight step 1 screening obtain pure TMX-13 bacterial strain, thallus by centrifugation (5000rpm,
15min, 4 DEG C) and washed three times with sterile phosphate buffer (PBS, pH 7.0), then it is resuspended with PBS into TMX-13 bacteria suspension
(OD600=1.0 or so)；The Diacloden of 2 μ g/mL is added as sole carbon in TMX-13 bacteria suspension by 1% bacterium amount that connects by volume again
It is cultivated 2,6,12,36,48,5 hours in the inorganic salt liquid culture medium of nitrogen source；Finally use high performance liquid chromatography tandem mass spectrometry
(LC-MS/MS) the Diacloden residual content in inorganic salt liquid culture medium is detected, the control group that comparison does not connect bacterium (is added 1%
PBS), the degradation rate of TMX-13 bacterial strain is calculated:
Diacloden degradation rate=(control group Diacloden content-experimental group Diacloden content)/control group Diacloden content *
100%；The final Diacloden degradation property for determining Enterobacter mori sp.TMX-13.
Above-mentioned high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) is referring to document: Li Y, Long L, Yan H, Ge J,
Cheng J,Ren L,Yu X,2018.Comparison of uptake,translocation and accumulation
of several neonicotinoids in komatsuna(Brassica rapa var.perviridis)from
contaminated soils.Chemosphere 2008,603-611.
For testing result as shown in Fig. 2, in Fig. 2, curve A is experimental group, and curve B is control group.It can be seen that the TMX- of 1% volume
The half-life period for the Diacloden that concentration is 2 μ g/mL can be reduced to 39 hours by 13 bacterium solutions from 78 hours, and degradation rate was at 48 hours
When reach 65%.Enterobacter mori sp.TMX-13 is sufficiently demonstrated to the efficient degradation characteristic of Diacloden.
The preparation of 2 TMX-13 microbial inoculum of embodiment
In the present embodiment, TMX-13 microbial inoculum obtains as follows:
A the bacterial strain TMX-13 that embodiment 1 obtains) is seeded to LB culture medium, 30 DEG C of scribing line choose single colonie culture twice
Afterwards, picking single bacterium is fallen in strain activation and culture base, 30 DEG C, 150-220rpm shaking table shake culture 12-24h, obtains activation bacterium
Kind；
B it) is inoculated with activated spawn to the fermentor equipped with seed culture medium, inoculum concentration is the 1% of seed culture medium volume,
25-38 DEG C, blowing air culture 16-24h obtains liquid seeds；
C liquid seeds) are inoculated with to the fermentor equipped with seed culture medium, inoculum concentration is the 1% of seed culture medium volume,
30-35 DEG C, it is protected from light culture under 200rpm to logarithmic growth phase, obtains thalline culture living；
D it) takes thalline culture 50ml living to be centrifuged 20min under 4 DEG C, 5000rpm, takes precipitating thallus sterile saline
It rinses 3 times, then is adjusted to bacterium solution OD with sterile saline600=1 (bacterial concentration about 107-8Cfu/mL), that is, TMX-13 is obtained
Microbial inoculum.
3 TMX-13 microbial inoculum pouring root of embodiment connects bacterium and repairs the pollution experiment of leaf vegetables Diacloden
Farmland natural soils are sampled, is first exposed to the sun air-dried, excessive stone grain is removed after 30 mesh sieve, soil constituent property is
PH 5.97, content of organic matter 66.8g/kg dry ground, total nitrogen content 0.36%, total carbon content 49.61%, total hydrogen content
5.7174%, total sulfur content 0.01%, oxygen content 44.37%, clay content 2.33%, mud content 16.1%, Sha Hanliang
81.5%.
After the root of the Xiaoqinling Nature Reserve Brassica rapa L of three leaves wholeheartedly to be all immersed in the TMX-13 microbial inoculum 4h that embodiment 2 obtains, move
It plants to above-mentioned farmland natural soils, control group is immersed in physiological saline；After cultivating 21d Brassica rapa L maturation, in Shanghai leafiness
Face sprays Diacloden pesticide (purchased from Jiangsu Hui Feng Biology Agriculture Co., Ltd, spraying by recommended dose), takes leaf after three days
Dish edible portion is remained with the Diacloden of high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) detection leaf vegetables edible portion
Content (detection method reference literature: Li Y, Long L, Yan H, Ge J, Cheng J, Ren L, Yu X,
2018.Comparison of uptake,translocation and accumulation of several
neonicotinoids in komatsuna(Brassica rapa var.perviridis)from contaminated
soils.Chemosphere 2008,603-611.)。
The experimental result is as shown in Figure 3, it is seen then that Diacloden residual content is reduced to from 1.52mg/Kg (control group 1)
0.62mg/Kg (experimental group 1), it was demonstrated that TMX-13 microbial inoculum can significantly reduce the Diacloden residual of leaf vegetables edible portion.
4 TMX-13 microbial inoculum foliage-spray of embodiment connects bacterium and repairs the pollution experiment of leaf vegetables Diacloden
Xiaoqinling Nature Reserve Brassica rapa L is planted into the farmland natural soils described in embodiment 3, after planting 40 days or so Brassica rapa L maturations,
It (purchased from Jiangsu Hui Feng Biology Agriculture Co., Ltd, sprays by recommended dose) in Brassica rapa L foliage-spray Diacloden pesticide,
After the pesticide on blade face is completely dry, the TMX-13 microbial inoculum that embodiment 2 obtains is sprayed on Brassica rapa L blade face, and (amount of spraying is about
60ml/m2), physiological saline is sprayed according to group, leaf vegetables edible portion is taken after three days, with high performance liquid chromatography tandem mass spectrometry (LC-
MS/MS) detect the Diacloden residual content of leaf vegetables edible portion (detection method is with embodiment 3).
The experimental result is as shown in figure 3, the microbial inoculum can remain the Diacloden of leaf vegetables edible portion from 1.50mg/Kg
(control group 2) is reduced significantly to 0.59mg/Kg (experimental group 2).
5 TMX-13 microbial inoculum pouring root of embodiment connects effect of the bacterium to leaf vegetables growth-promoting
After the root of the Xiaoqinling Nature Reserve Brassica rapa L of three leaves wholeheartedly is all immersed in the TMX-13 microbial inoculum that embodiment 2 obtains for 24 hours,
Transplanting to farmland natural soils, control group described in embodiment 3 is immersed in physiological saline；3 repetitions of every group of setting；Cultivate 21d
After Brassica rapa L is mature, extracts whole plant and observes growing state, experimental result is as shown in table 1 and Fig. 4:
Table 1
As it can be seen that TMX-13 can allow plant chlorophyll and biomass to increase, root system is more flourishing, and fibrous root significantly increases.It proves
TMX-13 microbial inoculum has the function of Brassica rapa L plant to promote growth.
Above-described embodiment demonstrates Diacloden in the degradable plant body of TMX-13 microbial inoculum and remains, and has growth-promoting to Brassica rapa L
Long effect.
Claims (7)
1. mulberry enterobacteria that one plant of deposit number is CGMCC NO:16236 (Enterobacter mori), which is straight
It is rod-shaped, no endogenous spore.
2. application of the mulberry enterobacteria as described in claim 1 in degradation Diacloden.
3. mulberry enterobacteria as described in claim 1 is promoting the application in leaf vegetables plant strain growth.
4. a kind of microbial inoculum containing mulberry enterobacteria as described in claim 1.
5. application as claimed in claim 2, which is characterized in that the application refers to that mulberry enterobacteria is in degradation leaf vegetables plant body
Application in interior Diacloden.
6. application as claimed in claim 5, which is characterized in that the application refers to, the mulberry enterobacteria is prepared into bacterium and is contained
Amount is 107-8The microbial inoculum of CFU/ml carries out leaching root or foliage-spray to leaf vegetables plant, to remaining thiophene in leaf vegetables plant of degrading
Worm piperazine.
7. application as claimed in claim 2, which is characterized in that the application refers to, the mulberry enterobacteria is prepared into bacterium and is contained
Amount is 107-8The microbial inoculum of CFU/ml carries out leaching root processing to leaf vegetables plant, to promote leaf vegetables plant strain growth.
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|---|---|---|---|---|
| CN101401991A (en) * | 2008-11-06 | 2009-04-08 | 浙江大学 | Uses of enterobacter aerogenes in degrading pyrethroid pesticide residue and preparation thereof |
| CN106479916A (en) * | 2016-09-29 | 2017-03-08 | 普洱学院 | A kind of enterobacter cloacae bacterial strain and its application |
| CN107354112A (en) * | 2017-08-23 | 2017-11-17 | 江苏南资环保股份有限公司 | One plant of thiophene worm aldehyde degradation bacteria and its application |
| CN107418918A (en) * | 2017-08-23 | 2017-12-01 | 江苏南资环保股份有限公司 | One plant of clothianidin degradation bacteria and its application |
| CN107699523A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | One plant of can degrade insecticide Buprofezin and the bacterium of Biphenthrin and its microbial inoculum of production |
-
2019
- 2019-06-18 CN CN201910525694.3A patent/CN110172430B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101401991A (en) * | 2008-11-06 | 2009-04-08 | 浙江大学 | Uses of enterobacter aerogenes in degrading pyrethroid pesticide residue and preparation thereof |
| CN106479916A (en) * | 2016-09-29 | 2017-03-08 | 普洱学院 | A kind of enterobacter cloacae bacterial strain and its application |
| CN107354112A (en) * | 2017-08-23 | 2017-11-17 | 江苏南资环保股份有限公司 | One plant of thiophene worm aldehyde degradation bacteria and its application |
| CN107418918A (en) * | 2017-08-23 | 2017-12-01 | 江苏南资环保股份有限公司 | One plant of clothianidin degradation bacteria and its application |
| CN107699523A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | One plant of can degrade insecticide Buprofezin and the bacterium of Biphenthrin and its microbial inoculum of production |
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